André Schützenmeister

Specification of Cortical Parenchyma and Stele of Maize Primary Roots by Asymmetric Levels of Auxin, Cytokinin, and Cytokinin-Regulated Proteins

In transverse orientation, maize (Zea mays) roots are composed of a central stele that is embedded in multiple layers of cortical parenchyma. The stele functions in the transport of water, nutrients, and photosynthates, while the cortical parenchyma fulfills metabolic functions that are not very well characterized. To better understand the molecular functions of these root tissues, protein- and phytohormone-profiling experiments were conducted. Two-dimensional gel electrophoresis combined with electrospray ionization tandem mass spectrometry identified 59 proteins that were preferentially accumulated in the cortical parenchyma and 11 stele-specific proteins. Hormone profiling revealed preferential accumulation of indole acetic acid and its conjugate indole acetic acid-aspartate in the stele and predominant localization of the cytokinin cis-zeatin, its precursor cis-zeatin riboside, and its conjugate cis-zeatin O-glucoside in the cortical parenchyma. A root-specific β-glucosidase that functions in the hydrolysis of cis-zeatin O-glucoside was preferentially accumulated in the cortical parenchyma. Similarly, four enzymes involved in ammonium assimilation that are regulated by cytokinin were preferentially accumulated in the cortical parenchyma. The antagonistic distribution of auxin and cytokinin in the stele and cortical parenchyma, together with the cortical parenchyma-specific accumulation of cytokinin-regulated proteins, suggest a molecular framework that specifies the function of these root tissues that also play a role in the formation of lateral roots from pericycle and endodermis cells.

Nonadditive protein accumulation patterns in Maize (Zea mays L.) hybrids during embryo development.

Heterosis describes the superior performance of heterozygous F(1)-hybrid plants compared to their homozygous parental inbred lines. In the present study, heterosis was detected for length, weight, and the time point of seminal root primordia initiation in maize (Zea mays L.) embryos of the reciprocal F(1)-hybrids UH005xUH250 and UH250xUH005. A two-dimensional gel electrophoresis (2-DE) proteome survey of the most abundant proteins of the reciprocal hybrids and their parental inbred lines 25 and 35 days after pollination revealed that 141 of 597 detected proteins (24%) exhibited nonadditive accumulation in at least one hybrid. Approximately 44% of all nonadditively accumulated proteins displayed an expression pattern that was not distinguishable from the low parent value. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analyses and subsequent functional classification of the 141 proteins revealed that development, protein metabolism, redox-regulation, glycolysis, and amino acid metabolism were the most prominent functional classes among nonadditively accumulated proteins. In 35-day-old embryos of the hybrid UH250xUH005, a significant up-regulation of enzymes related to glucose metabolism which often exceeded the best parent values was observed. A comparison of nonadditive protein accumulation between rice and maize embryo data sets revealed a significant overlap of nonadditively accumulated proteins suggesting conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis.

Comparative Transcriptome Profiling of Maize Coleoptilar Nodes during Shoot-Borne Root Initiation

A microarray analysis reveals 828 unique transcripts with expression related to crown root initiation during coleoptilar node development. Maize (Zea mays) develops an extensive shoot-borne root system to secure water and nutrient uptake and to provide anchorage in the soil. In this study, early coleoptilar node (first shoot node) development was subjected to a detailed morphological and histological analysis. Subsequently, microarray profiling via hybridization of oligonucleotide microarrays representing transcripts of 31,355 unique maize genes at three early stages of coleoptilar node development was performed. These pairwise comparisons of wild-type versus mutant rootless concerning crown and seminal roots (rtcs) coleoptilar nodes that do not initiate shoot-borne roots revealed 828 unique transcripts that displayed RTCS-dependent expression. A stage-specific functional analysis revealed overrepresentation of “cell wall,” “stress,” and “development”-related transcripts among the differentially expressed genes. Differential expression of a subset of 15 of 828 genes identified by these microarray experiments was independently confirmed by quantitative real-time-polymerase chain reaction. In silico promoter analyses revealed that 100 differentially expressed genes contained at least one LATERAL ORGAN BOUNDARIES domain (LBD) motif within 1 kb upstream of the ATG start codon. Electrophoretic mobility shift assay experiments demonstrated RTCS binding for four of these promoter sequences, supporting the notion that differentially accumulated genes containing LBD motifs are likely direct downstream targets of RTCS.

Tissue specific control of the maize (Zea mays L.) embryo, cortical parenchyma, and stele proteomes by RUM1 which regulates seminal and lateral root initiation.

The different root types of maize (Zea mays L.) originate from distinct tissues during development. The maize mutant rum1 (rootless with undetectable meristems 1) does not initiate seminal roots and lateral roots in the primary root. While seminal roots are laid down during embryogenesis, endodermis cells of the parenchyma, and pericycle cells of the stele contribute to the postembryonic initiation of lateral roots. In this study, tissue specific protein profiles of immature embryo, cortical parenchyma which includes endodermis, cortex and epidermis cell layers, and stele tissues were compared between wild-type and rum1 via 2-DE. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified 86 proteins encoded by 69 genes that were differentially accumulated between wild-type and rum1 (Fc>or=2; FDR<10%) demonstrating that RUM1 affects the proteome composition of cortical parenchyma, stele and embryo tissues. While several protein isoforms, protein families or members of biochemical pathways regulated by RUM1 were differentially accumulated in at least two tissues, other proteins displayed tissue specific expression differences. Multiple members of the globulin gene family displayed, for example, embryo specific expression differences, while different glycolysis related enzymes were differentially expressed in all three analyzed tissues. Proteins related to signal transduction and cell fate were overrepresented in cortical parenchyma versus embryo and embryo versus stele tissues, respectively, and might imply tissue specific functions of these protein classes.

Regulation of the maize (Zea mays L.) embryo proteome by RTCS which controls seminal root initiation

Seminal roots are initiated at the scutellar node during maize (Zea mays L.) embryo development. The maize mutant rtcs (rootless concerning crown and seminal roots) does not initiate seminal roots while its wild-type siblings form on average 2.9 seminal roots per seedling. In this study, proteome profiles of 25-day-old immature maize embryos were compared between wild-type and rtcs plants via two-dimensional electrophoresis (2-DE). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified 23 proteins encoded by 21 different genes that were differentially accumulated between wild-type and rtcs embryos (Fc> or =2; FDR<10%). Among the differentially accumulated proteins, two isoforms of a phosphoglycerate kinase and a malate dehydrogenase were preferentially accumulated in wild-type embryos. Both enzymes are related to the generation of energy-rich ATP or NADPH molecules and are crucial checkpoints of cellular energetics in plants. Comparison of embryonic proteins differentially accumulated between wild-type and rtcs embryos revealed little overlap with proteins differentially accumulated between wild-type and rum1 embryos which also do not initiate seminal roots. This might be due to distinct influences of RTCS and RUM1 on the composition of the embryo proteome, but could also be explained by different stages of embryo development that were analyzed in these studies.

Residual analysis of linear mixed models using a simulation approach

In the framework of the general linear model, residuals are routinely used to check model assumptions, such as homoscedasticity, normality, and linearity of effects. Residuals can also be employed to detect possible outliers. Various types of residuals may be defined for linear mixed models. It is shown how residual plots can be used to check model assumptions by comparing empirical residual distributions with appropriate null distributions based on a parametric bootstrap approach. This allows constructing simultaneous tolerance bounds, which helps in assessing the normality and homoscedasticity of residuals of linear mixed models, identifying possible outliers and interpreting residual plots. The usefulness of this method is demonstrated by applying it to several previously published datasets.

Background correction of two-colour cDNA microarray data using spatial smoothing methods.

The analysis of two-colour cDNA microarray data usually involves subtracting background values from foreground values prior to normalization and further analysis. This approach has the advantage of reducing bias and the disadvantage of blowing up the variance of lower abundant spots. Whenever background subtraction is considered, it implicitly assumes locally constant background values. In practice, this assumption is often not met, which casts doubts on the usefulness of simple background subtraction. In order to improve background correction, we propose local background smoothing within the pre-processing pipeline of cDNA microarray data prior to background correction. For this purpose, we employ a geostatistical framework with ordinary kriging using both isotropic and anisotropic models of spatial correlation and 2-D locally weighted regression. We show that application of local background smoothing prior to background correction is beneficial in comparison to using raw background estimates. This is done using data of a self-versus-self experiment in Arabidopsis where subsets of differentially expressed genes were simulated. Using locally smoothed background values in conjunction with existing background correction methods increases the power, increases the accuracy and decreases the number of false positive results.